Ketone body metabolism and sleep homeostasis in mice

Neuropharmacology 79 (2014) 399e404

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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Ketone body metabolism and sleep homeostasis in mice Sachiko Chikahisa, Noriyuki Shimizu, Tetsuya Shiuchi, Hiroyoshi Séi* Department of Integrative Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 September 2013 Received in revised form 5 December 2013 Accepted 6 December 2013

A link has been established between energy metabolism and sleep homeostasis. The ketone bodies acetoacetate and b-hydroxybutyrate, generated from the breakdown of fatty acids, are major metabolic fuels for the brain under conditions of low glucose availability. Ketogenesis is modulated by the activity of peroxisome proliferator-activated receptor alpha (PPARa), and treatment with a PPAR activator has been shown to induce a marked increase in plasma acetoacetate and decreased b-hydroxybutyrate in mice, accompanied by increased slow-wave activity during non-rapid eye movement (NREM) sleep. The present study investigated the role of ketone bodies in sleep regulation. Six-hour sleep deprivation increased plasma ketone bodies and their ratio (acetoacetate/b-hydroxybutyrate) in 10-week-old male mice. Moreover, sleep deprivation increased mRNA expression of ketogenic genes such as PPARa and 3-hydroxy-3-methylglutarate-CoA synthase 2 in the brain and decreased ketolytic enzymes such as succinyl-CoA: 3-oxoacid CoA transferase. In addition, central injection of acetoacetate, but not bhydroxybutyrate, markedly increased slow-wave activity during NREM sleep and suppressed glutamate release. Central metabolism of ketone bodies, especially acetoacetate, appears to play a role in the regulation of sleep homeostasis. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Sleep Ketone bodies Slow-wave activity PPARa

1. Introduction Ketone bodies become major fuels in most tissues during starvation, prolonged exercise, or consumption of a high-fat, lowcarbohydrate diet (Robinson and Williamson, 1980). Conditions of reduced glucose availability lead to increased ketone production (ketogenesis) and use. Ketones, such as acetoacetate (AcAc) and bhydroxybutyrate (BHB), are generated from the breakdown of fatty acids (Fukao et al., 2004; Robinson and Williamson, 1980). Circulating levels of ketone bodies are determined by their rates of production and utilization (ketolysis). Although the liver is generally believed to be the major organ that supplies the brain with ketone bodies, it has been reported that astrocytes can also produce ketone bodies from fatty acids under conditions of glucose deprivation (Auestad et al., 1991; Blazquez et al., 1998). Sequential ketogenic reactions catalyzed by mitochondrial thiolase, 3-hydroxy-3-methylglutarate-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA (HMG-CoA) lyase convert acetyl-CoA (Ac-CoA) to the ketone body AcAc (Cullingford, 2004; Fukao et al., 2004; Hegardt, 1999). AcAc can be reversibly reduced to

* Corresponding author. Tel.: þ81 88 633 7057; fax: þ81 88 633 9251. E-mail address: [email protected] (H. Séi). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.12.009

BHB by mitochondrial BHB dehydrogenase (HBD) in an NADþ/ NADH-coupled redox reaction (Hegardt, 1999). The ratio of AcAc to BHB reflects the redox state within the mitochondrial matrix (Constantin et al., 2011; Katsuyama et al., 1999). In extrahepatic tissues, AcAc is activated to acetoacetyl-CoA (AcAc-CoA) by the mitochondrial matrix enzyme succinyl-CoA-3-oxoacid CoA transferase (SCOT), a mitochondrial CoA transferase in mammals (Fukao et al., 2004; Laffel, 1999; Williamson et al., 1971). Ac-CoA produced by the action of AcAc-CoA thiolase enters the tricarboxylic acid (TCA) cycle for terminal oxidation and provides fuel for ATP synthesis (Fukao et al., 2004; Laffel, 1999; Robinson and Williamson, 1980). The relationship between ketone body metabolism in the brain and neuronal activity continues to be investigated, and many mechanisms of ketone body action have been suggested (Cullingford, 2004; Guzman and Blazquez, 2001; Morris, 2005; Nehlig, 2004). In recent years, a considerable number of studies using normal and obese or diabetic animals have shown that sleep/wake regulation and energy metabolism are closely intertwined (Laposky et al., 2008; Martins et al., 2008). High-fat feeding and food restriction paradigms, both of which are believed to enhance ketogenesis, can affect sleep/wake patterns (Alvarenga et al., 2005; Jenkins et al., 2006). In addition, a recent study in humans showed evidence that suppression of stages 3 and 4 of non-rapid

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eye movement (NREM) sleep, which are accompanied by reduced slow-wave activity (SWA, power density of the electroencephalogram (EEG) delta band between 0.5 and 2.0 Hz in humans), resulted in decreased insulin sensitivity and glucose tolerance, and the risk of type 2 diabetes was therefore increased (Tasali et al., 2008). SWA in NREM sleep is believed to be a variable of sleep depth and homeostatic need for sleep because it is enhanced after sleep deprivation (SD) (Borbely et al., 1981; Dijk et al., 1990). Since ketone bodies are elevated in patients with diabetes, and conversely, treatment with ketone bodies was shown to improve insulin sensitivity in type 2 diabetic rats (Laffel, 1999; Park et al., 2011), disturbances in SWA are likely related to metabolic impairment due to alteration of ketogenesis and/or ketolysis. Thus, ketone bodies may be involved in sleep/wake regulation, but this has never been studied. Ketogenesis is modulated by the activity of peroxisome proliferator-activated receptor alpha (PPARa), one of three PPAR subtypes (a, b, and g). PPARa is expressed in the liver, muscle, and brain, and controls transcription of many genes involved in fatty acid metabolism including those involved in ketogenesis in response to fasting (Cullingford, 2004; Desvergne and Wahli, 1999). We recently demonstrated that treatment with bezafibrate, a PPAR agonist, increased SWA in NREM sleep in mice over 24 h, accompanied by increased expression of genes encoding ketogenic enzymes such as Hmgcs2 in the liver (Chikahisa et al., 2008). In that study, bezafibrate-treated mice showed increased AcAc and decreased BHB in plasma, resulting in a very high ketone body ratio (AcAc/BHB). However, it was not clear whether ketone body metabolism in the brain influenced sleep homeostasis. In this article, we investigated the role of ketone body metabolism in sleep homeostasis by measuring plasma ketone body levels in sleep-deprived mice, and by evaluating SWA during NREM sleep in mice centrally injected with ketone bodies. 2. Materials and methods 2.1. Animals All experiments were performed using male Jcl/ICR mice (Slc Inc., Shizuoka, Japan). Eight-week-old mice were fed ad libitum and maintained on a 12-h lighte dark (L/D) cycle (lights on at 0900) at a controlled ambient temperature (23  1  C). The Animal Study Committee of Tokushima University approved these experiments, and we performed them in accordance with Guidelines for the Care and Use of Animals approved by the Council of the Physiological Society of Japan.

2.4. Pharmacological treatments and injection procedure Cannulae were implanted intracerebroventricularly (icv) at the time of surgery for EEG/EMG, as previously described (Chikahisa et al., 2009). Ketone bodies, including AcAc (lithium acetoacetate, Sigma Chemical Co., St. Louis, MO, USA) and BHB (sodium 3-hydroxybutyrate, Sigma Chemical Co.), were injected (icv) into 10week-old mice at ZT0 or ZT12. AcAc and BHB (50 mg and 200 mg) were dissolved in 1.0 ml Ringer solution and injected slowly over 1 min using a Hamilton microsyringe. 2.5. Real-time RT-PCR analysis Tissues used for molecular analysis were dissected immediately after decapitation, frozen in liquid nitrogen, and stored at 80  C until use. Mice were euthanized at ZT6 and ZT12. Tissue preparation and analysis were performed as previously described (Chikahisa et al., 2008). We used pre-designed, gene-specific TaqMan probes and primer sets (Applied Biosystems, Foster City, CA) to assess expression of the following genes: Ppara (Mm00440939_m1), Hmgcs2 (Mm00520236_m1), Scot (Mm00499303_m1), and acetoacetyl-CoA synthetase (Aacs; Mm00513427_m1, a cytosol ketolysis enzyme which catalyzes the synthesis of acetoacetyl-CoA from AcAc). Real-time RT-PCR was carried out using an Applied Biosystems 7900HT real-time RT-PCR system and TaqMan universal PCR Master Mix (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. For endogenous quantity control, we normalized values to those for the housekeeping gene b-actin (Mm00607939_s1). 2.6. Measurement of plasma ketone body and glucose levels Trunk blood was collected from each group at the time of decapitation for realtime RT-PCR analysis. The plasma ketone bodies AcAc and BHB were measured enzymatically using an automatic analyzer system (JCA-BM12; JEOL, Tokyo, Japan) and reagents for enzymatic measurement of ketone bodies (Kainos Laboratories, Tokyo, Japan). Glucose was detected using a glucose biosensor (LifeScan, Inc., Milpitas, CA, USA). 2.7. In vivo microdialysis The microdialysis cannula was implanted into the left lateral ventricle (AP -0.5 mm; ML 1.2 mm; V 1.5 mm to bregma), and the cannula for drug injection was implanted obliquely into the right lateral ventricle (AP -2.2 mm; ML 0.9 mm; V 2.5 mm, Angle 30 relative to bregma) of mice under general anesthesia with a cocktail of ketamine (100 mg/kg) and xylazine (25 mg/kg). After 2 weeks of recovery, a microdialysis probe with a 2 mm-long semipermeable membrane (Eicom, Kyoto, Japan) was inserted into the lateral ventricle 3e4 h prior to the experiment. The microdialysis lines were continuously perfused with Ringer’s solution at a rate of 1 ml/min. Dialysate was collected from conscious mice every 30 min from 60 min before to 150 min after drug injection. After sampling the baseline for 60 min, AcAc and BHB (200 mg, icv) were injected into control (non-sleep deprived) mice at ZT6. Glutamate content in the cerebrospinal fluid was determined using an Eicom high-performance liquid chromatography-electrochemical detector (HPLC-ECD) system. Dialysate samples (30 ml) were injected onto a column (Eicompak SC-5ODS, 150 mm  3.0 mm i.d.) with a pre-column (CA-ODS, 4 mm  3.0 mm i.d.). The glutamate was eluted with 0.1 M potassium phosphate at a flow rate of 0.5 ml/min, post-labeled with o-phthalaldehyde under alkaline conditions, and detected with an ECD-300 detector (Eicom, Kyoto, Japan).

2.2. Sleep recording and analysis

2.8. Statistics

EEG/electromyogram (EMG) implantation surgery for sleep recording and telemetry (TA10TA-F20; Data Sciences Int., USA) for recording of body temperature and locomotor activity were performed as previously described (Chikahisa et al., 2009). Off-line sleep scoring was done on the computer screen by visual assessment of EEG and EMG activity using the Spike2 analysis program (CED, Cambridge, UK). Vigilance states were based on data binned in 6-s epochs and classified as wakefulness, rapid eye movement (REM) or NREM sleep. The EEG power spectrum in the epoch determined to represent NREM sleep was calculated by Fast Fourier Transform using the Spike2 analysis program. The EEG delta frequency band was set at 0.5e4.0 Hz. The delta power was normalized as a percentage of the total power (0.5e50 Hz). Body temperature, locomotor activity, time spent sleeping and awake, and EEG delta power were averaged for hourly intervals.

Results are expressed as means  SEM. Plasma ketone bodies and glucose levels were analyzed using Student’s t test. Real-time RT-PCR data were analyzed using one-way analysis of variance (ANOVA) followed by Scheffe’s post-hoc test. Changes in sleep architecture, SWA, body temperature, and locomotor activity were analyzed by repeated measures of two-way ANOVA followed by Scheffe’s post-hoc test. p < 0.05 was assumed to indicate statistical significance.

2.3. Procedure for SD and food deprivation (FD) Ten-week old mice were sleep-deprived for 6 h between Zeitgeber Time (ZT) 0 and ZT6 using a small soft brush to touch the back of the mouse several times when it appeared to become sleepy. At ZT6, SD ended and half of the sleep-deprived mice were euthanized, while the other half had a 6-h period of uninterrupted recovery sleep and were euthanized at ZT12. The control (non-SD) mice were also euthanized at ZT6 and ZT12. For FD, food pellets were carefully removed from the recording cages at the onset of the light period (ZT0), and returned again to the cage at ZT6.

3. Results 3.1. SD increases blood ketone bodies Six-hour SD significantly increased NREM sleep and SWA during subsequent NREM sleep in mice (Fig. S1). Based on this general finding and our previous findings (Chikahisa et al., 2008), we hypothesized that SD would alter ketone body levels, inducing the homeostatic response to sleep loss. Six-hour SD induced a marked increase in both plasma AcAc and BHB (Fig. 1A and B; ZT6). Ketone body ratio was also increased because the rise in AcAc was greater than that of BHB after SD (Fig. 1D; ZT6). These changes in ketone body content returned to control levels after a 6-h recovery period

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Fig. 1. Sleep deprivation (SD) for 6 h increased plasma ketone body levels. Plasma concentrations of acetoacetate (AcAc)(A), b-hydroxybutyrate (BHB)(B), total ketones (C), ketone body ratio (D), and glucose (E) in mice euthanized immediately after 6-h SD (ZT6) or the subsequent 6-h recovery period (ZT12). Open bars indicate control non-SD mice, and closed bars indicate SD mice. Another group of mice were subjected to 6-h food deprivation (FD); their data are shown as gray bars (AeE). All data are expressed as means  SEM (n ¼ 6/ group). *p < 0.05, **p < 0.01, control versus SD.

(ZT12). Food intake during SD was slightly higher than that of control mice, but this did not reach statistical significance (Fig. S2). We also prepared another group of mice for 6-h FD during the same time period as SD. Although 6-h FD greatly increased both AcAc and BHB, it did not affect their relative ratio (Fig. 1A, B and D).

while BHB injection did not affect it (Fig. S3). These results suggest that central AcAc is an enhancer of SWA during NREM sleep. Furthermore, icv injection of AcAc at a low dose decreased body temperature (Fig. 4E), while locomotor activity was not changed by either AcAc or BHB injection (Fig. 4F).

3.2. SD activates ketogenesis in the brain

3.4. Effect of ketone bodies on glutamate release

To determine the effects of SD on ketone body metabolism in the brain, we measured expression of genes related to ketogenesis and ketolysis in the hypothalamus and cortex. Six-hour SD significantly increased mRNA expression of Ppara (a ketogenic transcription factor) and its downstream target gene Hmgcs2 (a rate-limiting enzyme for ketogenesis) in the hypothalamus compared with that of control or 6-h FD mice (Fig. 2A; ZT6). In the cortex, SD also increased Ppara expression, but significantly decreased mRNA expression of the ketolytic enzyme genes Scot and Aacs compared with control or 6-h FD mice (Fig. 2B; ZT6). These changes in gene expression in the brain returned to control levels after a 6h recovery period (Fig. 2A and B; ZT12), suggesting that SD affects ketone body metabolism in the brain. In contrast, SD for 6 h did not affect mRNA expression of Ppara and Hmgcs2 in the liver, while 6h FD tended to increase liver Ppara mRNA (Fig. 3).

Circulating AcAc can suppress the activity of vesicular glutamate transporters (VGLUTs) leading to decreased glutamate release and consequent suppression of excitatory neurotransmission (Juge et al., 2010). Glutamate is well known to be associated with sleep/wake regulation and cortical projections of the glutamatergic system are important for cortical activation and wakefulness (Brown et al., 2012). We therefore investigated whether icv injection of AcAc would suppress glutamate release using in vivo microdialysis. Data on SWA in mice centrally injected with AcAc were fully consistent with the microdialysis finding that glutamate release in the lateral ventricle immediately decreased after injection of AcAc but not of BHB (Fig. 5).

3.3. Effects of ketone bodies on sleep regulation Sleep-deprived mice, which showed increased SWA during NREM sleep, have a significantly increased plasma ketone body content and ratio. To investigate whether AcAc or BHB is more important for regulation of sleep, AcAc or BHB was injected into the brains of mice at ZT12. Interestingly, icv injection of AcAc significantly increased SWA during NREM sleep in a dose-dependent manner (Fig. 4D), accompanied by a slight decrease in the amount of REM sleep (Fig. 4C). In contrast, BHB injection at ZT12 affected neither SWA nor sleep/wake patterning (Fig. 4AeD). Icv injection of AcAc at ZT0 also enhanced SWA during NREM sleep,

4. Discussion The principal findings of this study are that prolonged wakefulness activates brain ketogenesis, and that central injection of AcAc enhances SWA during NREM sleep and attenuates glutamate release in the brain (Fig. S4). PPARa, a key regulator of the lipid metabolism pathway, stimulates ketogenesis and lipid b-oxidation in mitochondria (Cullingford, 2004; Desvergne and Wahli, 1999). In our previous study, mice treated with bezafibrate, a pan-PPAR agonist, showed increased plasma AcAc and an increased ketone body ratio with increased SWA in NREM sleep (Chikahisa et al., 2008). In the present study, mice deprived of sleep for 6 h showed increased expression of the Ppara gene and its downstream target gene Hmgcs2 in the hypothalamus and cortex. In addition, 6-h SD, which

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Fig. 2. Expression of genes related to regulation of ketone body metabolism in the brain was affected by sleep deprivation (SD). Expression of Ppara, Hmgcs2, Scot, and Aacs mRNA was measured in the hypothalamus (A) and cortex (B) of mice euthanized immediately after 6-h SD/food deprivation (FD) (ZT6) or the subsequent 6-h recovery period (ZT12). Open bars indicate control mice, closed bars indicate SD mice, and gray bars indicate FD mice. All data are expressed as means  SEM (n ¼ 6e7/group). *p < 0.05, **p < 0.01, control versus SD. #p < 0.05, ##p < 0.01, SD versus FD.

increases SWA in subsequent sleep, markedly increased plasma ketone bodies and ketone body ratio. These results suggest that prolonged wakefulness activates brain ketogenesis, which would increase plasma ketone bodies and their ratio. With regard to changes in the ketone body ratio, decreased cortical mRNA expression of Scot, which catalyzes the mitochondrial rate-determining step in ketolysis, may be involved in the increased plasma ketone body ratio observed in SD mice. Scot activity is known to be down-regulated by high (>5 mM) intracellular levels of AcAc (Fenselau and Wallis, 1974). It has also been reported that neonatal Scot knockout (Oxct1/) mice showed an accumulation of plasma AcAc, resulting in a markedly increased peripheral ketone body ratio because of ketolytic lesions that interfere with the reaction catalyzed by Scot (Cotter et al., 2011). In the present study, FD for 6 h did not affect the plasma ketone body ratio, although it induced a larger increase in both AcAc and BHB levels than SD. The results of our previous study showed that FD itself has

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Fig. 3. Effects of sleep deprivation (SD) and food deprivation (FD) on hepatic expression of genes related to ketogenesis. Expression of Ppara and Hmgcs2 mRNA was measured in the livers of mice euthanized immediately after 6-h SD/FD (ZT6) or the subsequent 6-h recovery period (ZT12). Open bars indicate control mice, closed bars indicate SD mice, and gray bars indicate FD mice. All data are expressed as means  SEM (n ¼ 6e7/group).

little effect on SWA during NREM sleep (Shimizu et al., 2011). These data indicate that the effects of SD on ketone body metabolism are distinct from FD-induced ketogenesis. In order to investigate the direct effects of ketone bodies in the brain on sleep/wake regulation, AcAc and BHB were injected into the lateral ventricle in conscious mice. Icv injection of AcAc increased SWA during NREM sleep, while BHB injection did not show any significant effect, indicating that central AcAc is more important for sleep regulation than BHB. As to the central function of ketone bodies, recent work has suggested that ketone bodies compete with Cl -dependent action of VGLUTs to regulate loading of glutamate into presynaptic vesicles, and to suppress glutamate release in vivo (Juge et al., 2010). It has been demonstrated that Cl- is an allosteric activator of VGLUTs that is competitively inhibited by ketone bodies, and that AcAc acts on VGLUTs more potently than BHB (Juge et al., 2010). We also found in the present study that icv injection of AcAc decreased glutamate release in freely moving mice, while neither BHB nor vehicle injection affected it. Since glutamate is known to promote wakefulness (Brown et al., 2012), brain ketone bodies, and especially AcAc, may increase SWA in NREM sleep through the suppression of excitatory neuronal transmission due to suppression of glutamate release. In vivo studies in rodents and many clinical studies have suggested that ketone bodies have anticonvulsant and antiepileptic effects, because a ketogenic (high fat and low carbohydrate) diet and fasting have been shown to decrease seizures (Cullingford, 2004; Hartman et al., 2007; McNally and Hartman, 2012; Neal et al., 2009). Numerous mechanisms for this phenomenon have been proposed, including increased levels of the inhibitory neurotransmitter g-aminobutyric acid (GABA) in the brain (Dahlin et al., 2005; Wang et al., 2003; Yudkoff et al., 2001). Activation of GABAergic neurons in the ventrolateral preoptic area (VLPO) is well known to increase SWA in NREM sleep (Brown et al., 2012). The effect of ketone bodies on SWA observed in this study may be partly mediated by GABA signaling, although no significant differences in GABA levels measured by microdialysis were observed among the

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Vehicle Low (50 µg) High (200 µg) Fig. 4. Central injection of ketone bodies at the onset of the dark phase increased slow-wave activity (SWA) during non-rapid eye movement (NREM) sleep. Hourly time course for wakefulness (A), NREM sleep (B), and rapid eye movement (REM) sleep (C), SWA in NREM sleep (D), body temperature (E), and locomotor activity (F) after icv injection of vehicle, acetoacetate (AcAc) (left panels), or b-hydroxybutyrate (BHB) (right panels). Open circles indicate mice injected with vehicle, gray circles indicate a low dose (50 mg), and closed circles indicate a high dose (200 mg) of each drug. All data are expressed as means  SEM (n ¼ 6/group). *p < 0.05, **p < 0.01, vehicle versus high dose. ##p < 0.01, low versus high dose.

three icv injection groups (AcAc, BHB, and vehicle, Fig. S5). Other proposed mechanisms for the anticonvulsant effects of a ketogenic diet include the opening probability of ATP-sensitive Kþ (KATP) channels (Ma et al., 2007; Tanner et al., 2011), and adenosine A1 receptor-mediated neurotransmission (Masino et al., 2011). Ketone bodies can reduce firing rate by lowering cytoplasmic ATP, which was shown to activate KATP channels in slices from rat brains (Ma et al., 2007). In addition, ketone bodies have also been reported to suppress seizures by increasing adenosine A1 receptor-mediated inhibition (Masino et al., 2011). Like GABA, adenosine and its receptor are involved in sleep homeostasis, and SWA in NREM sleep displays a strong positive correlation with adenosine levels in the

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Time after injection (30 min for each circle ) Fig. 5. Central injection of AcAc decreased glutamate release. In the microdialysis experiment, glutamate release in the lateral ventricle was measured after icv injection of vehicle (open circles), b-hydroxybutyrate (BHB) (gray circles), or acetoacetate (AcAc) (closed circles). Each circle represents glutamate levels over 30 min. Data after drug injection (D1eD6) are expressed as the percentage change from the baseline value (B1 and B2) of each group. Arrow (Y) indicates the time of drug injection. All data are expressed as means  SEM (n ¼ 5e6/group). *p < 0.05, vehicle versus AcAc.

brain during the recovery period after SD (Porkka-Heiskanen and Kalinchuk, 2011). The neural and molecular mechanisms by which ketone bodies influence EEG delta oscillation observed in SWA during NREM sleep should be clarified in future studies. Because of technical limitations in the measurement of ketone bodies in mouse brains, we could not determine the origin of the change in peripheral plasma ketone bodies. Although most ketone bodies are produced in the liver, there is evidence that astrocytes can also produce ketone bodies from fatty acids (Auestad et al., 1991; Blazquez et al., 1998). In the present study, in fact, 6-h SD also increased expression of the ketogenic genes Ppara and Hmgcs2 in the brain, while it did not affect their expression in the liver. These results suggest that brain ketogenesis is more closely linked to sleep regulation than liver ketogenesis. AMP-activated protein kinase (AMPK) is known to play an important role in sustaining ketone body production in astrocytes under conditions of metabolic stress, such as hypoxia (Blazquez et al., 1999; Guzman and Blazquez, 2004). AMPK is activated by an increase in the AMP/ ATP ratio both directly and via activation of an upstream AMPK kinase such as Ca2þ/calmodulin-dependent protein kinase kinase b (CaMKK2) (Kahn et al., 2005; Kola, 2008). Once activated, AMPK phosphorylates and inactivates acetyl-CoA carboxylase (ACC), thereby decreasing malonyl-CoA concentration and allowing carnitine palmitoyltransferase 1 (CPT1) to be released from inhibition and thereby to increase the supply of fatty acid substrate (Kahn et al., 2005; Kola, 2008). Activation of AMPK during hypoxia provides a supply of ketone bodies from astrocytes to neurons via the cascade in order to compensate for depression of intramitochondrial fatty acid b-oxidation under low-oxygen conditions (Blazquez et al., 1999; Guzman and Blazquez, 2004). We previously

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observed that AMPK activity in the hypothalamus increases after SD, and is accompanied by increased Camkk2 mRNA expression (Chikahisa et al., 2009). SD induces a significant reduction in ATP content in the brain (Dworak et al., 2010), and ATP levels in the brain have been directly related to regulation of SWA in NREM sleep (Chikahisa and Sei, 2011). Prolonged wakefulness may activate central lipid metabolism through activation of AMPK resulting from an increased AMP/ATP ratio, thereby up-regulating ketogenesis. We are now planning to investigate whether SD enhances astroglial ketogenesis via AMPK activation. In addition, it is necessary to investigate the effects of central inhibition of ketogenesis and PPARa signaling on regulation of sleep homeostasis. We are now measuring sleep homeostasis in mice centrally injected with Hmgcs2 antisense oligonucleotides and PPARa antagonists such as GW6471. In conclusion, enhanced SWA during NREM sleep induced by SD was accompanied by increased plasma ketone bodies and ketogenic gene expression in the brain. Moreover, central injection of AcAc increased SWA via suppression of glutaminergic neurotransmission, suggesting that ketone bodies and their metabolism in the brain may play an important role in sleep homeostasis. Our findings provide additional evidence for an interaction of energy metabolism and sleep/wake regulation. Acknowledgments This study was supported by Grants-in-Aid for Young Scientists (B) (23730706) to S.C and (22590224) to H.S from the Japan Society for the Promotion of Science (JSPS). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2013.12.009 References Alvarenga, T.A., Andersen, M.L., Papale, L.A., Antunes, I.B., Tufik, S., 2005. Influence of long-term food restriction on sleep pattern in male rats. Brain Res. 1057, 49e 56. Auestad, N., Korsak, R.A., Morrow, J.W., Edmond, J., 1991. Fatty acid oxidation and ketogenesis by astrocytes in primary culture. J. Neurochem. 56, 1376e1386. Blazquez, C., Sanchez, C., Velasco, G., Guzman, M., 1998. Role of carnitine palmitoyltransferase I in the control of ketogenesis in primary cultures of rat astrocytes. J. Neurochem. 71, 1597e1606. Blazquez, C., Woods, A., de Ceballos, M.L., Carling, D., Guzman, M., 1999. The AMPactivated protein kinase is involved in the regulation of ketone body production by astrocytes. J. Neurochem. 73, 1674e1682. Borbely, A.A., Baumann, F., Brandeis, D., Strauch, I., Lehmann, D., 1981. Sleep deprivation: effect on sleep stages and EEG power density in man. Electroencephalogr. Clin. Neurophysiol. 51, 483e495. Brown, R.E., Basheer, R., McKenna, J.T., Strecker, R.E., McCarley, R.W., 2012. Control of sleep and wakefulness. Physiol. Rev. 92, 1087e1187. Chikahisa, S., Fujiki, N., Kitaoka, K., Shimizu, N., Sei, H., 2009. Central AMPK contributes to sleep homeostasis in mice. Neuropharmacology 57, 369e374. Chikahisa, S., Sei, H., 2011. The role of ATP in sleep regulation. Front Neurol. 2, 87. Chikahisa, S., Tominaga, K., Kawai, T., Kitaoka, K., Oishi, K., Ishida, N., Rokutan, K., Sei, H., 2008. Bezafibrate, a peroxisome proliferator-activated receptors agonist, decreases body temperature and enhances electroencephalogram deltaoscillation during sleep in mice. Endocrinology 149, 5262e5271. Constantin, R.P., Constantin, J., Pagadigorria, C.L., Ishii-Iwamoto, E.L., Bracht, A., de Castro, C.V., Yamamoto, N.S., 2011. Prooxidant activity of fisetin: effects on energy metabolism in the rat liver. J. Biochem. Mol. Toxicol. 25, 117e126. Cotter, D.G., d’Avignon, D.A., Wentz, A.E., Weber, M.L., Crawford, P.A., 2011. Obligate role for ketone body oxidation in neonatal metabolic homeostasis. J. Biol. Chem. 286, 6902e6910. Cullingford, T.E., 2004. The ketogenic diet; fatty acids, fatty acid-activated receptors and neurological disorders. Prostaglandins Leukot. Essent. Fatty Acids 70, 253e264. Dahlin, M., Elfving, A., Ungerstedt, U., Amark, P., 2005. The ketogenic diet influences the levels of excitatory and inhibitory amino acids in the CSF in children with refractory epilepsy. Epilepsy Res. 64, 115e125.

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